PROJECT SUMMARY Over the past several years, we have studied changes in critical blood-brain barrier (BBB) tight junction (TJ) proteins (i.e., claudin-5, occludin) in response to diseases or drugs. Our work has shown that claudin-5 and occludin expression and trafficking is modulated by pain (i.e., TJ dysregulation), an effect that increases brain uptake of opioids such as codeine. Our preliminary data show that acetaminophen (APAP) can modulate expression of claudin-5 at the BBB and increase paracellular permeability (i.e., ?leak?). Leak has previously been shown to occur with low dose, acute cocaine administration as well as methamphetamine. Both APAP and pain target transforming growth factor- ? (TGF-?) signaling, a pathway that controls TJ expression and BBB integrity. In this grant, we hypothesize that APAP, by itself and in the setting of pain, can increase BBB ?leak? and enhance CNS opioid delivery. These studies are highly significant because they will uncover mechanisms of altered opioid antinociception and adverse drug events that can occur in individuals who abuse or misuse opioids or APAP. Additionally, APAP is taken with other centrally acting drugs where ?APAP leak? can lead to therapeutic toxicity. Aim 1: To investigate, in vivo, changes in TJ protein expression and trafficking at the BBB following APAP administration. We will study how APAP alters CNS effects and adverse events of opioids. We will investigate the time course of changes in transmembrane TJ protein (i.e., claudin-5, occludin) expression and trafficking and the dose-response relationship of APAP on changes in TJ protein complexes in male and female Sprague-Dawley rats (Aim 1A). We will then study the temporal relationship between CNS opioid delivery, opioid-associated antinociception, respiratory depression, and opioid-associated reward behavior (Aim 1B). Since we have shown that transforming growth factor- ? (TGF-?) signaling regulates BBB integrity, we will study the role of this pathway on claudin-5 and occludin expression and trafficking (Aim 1C). Aim 2: To examine involvement of APAP on BBB integrity in pain. Using established in vivo pain models (i.e., l-carrageenan-induced acute inflammatory pain, chronic pain spinal nerve ligation), we will study the time course of APAP effects on TJ protein expression/trafficking (i.e., claudin-5, occludin) as well as on CNS opioid uptake (Aim 2A). We will demonstrate effects of single versus multiple doses of APAP on TGF-? signaling pathways in both pain models (Aim 2B). We will study how changes in CNS opioid delivery affect opioid antinociception, respiratory depression, and opioid-associated reward behavior (Aim 2C). Since we have shown that APAP increases functional expression of the critical opioid transporter P-glycoprotein, we will examine the contribution of claudin-5/occludin modulation and P-gp changes to CNS opioid delivery (Aim 2D). Our group is uniquely positioned to provide a mechanistic explanation (i.e., signaling, activity, trafficking) for adverse drug events in individuals who abuse/misuse prescription pain drugs.